Analysis of circulating microRNAs and their post-transcriptional

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Analysis of circulating microRNAs and their post-transcriptional modifications in cancer serum by on-line solid-phase extraction capillary electrophoresis-mass spectrometry Roger Pero-Gascon, Victoria Sanz-Nebot, Maxim V Berezovski, and Fernando Benavente Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b00405 • Publication Date (Web): 07 May 2018 Downloaded from http://pubs.acs.org on May 7, 2018

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Analytical Chemistry

Analysis of circulating microRNAs and their post-transcriptional modifications in cancer serum by on-line solid-phase extraction capillary electrophoresis-mass spectrometry

Roger Pero-Gascon1, Victoria Sanz-Nebot*1, Maxim V. Berezovski2, Fernando Benavente1 1

Department of Chemical Engineering and Analytical Chemistry, Institute for Research

on Nutrition and Food Safety (INSA·UB), University of Barcelona, Barcelona, Spain

2

Department of Chemistry and Biomolecular Sciences, University of Ottawa, Ottawa,

Ontario, Canada *

Corresponding author: [email protected] (V. Sanz-Nebot, PhD) Tel: (+34) 934021283, Fax: (+34) 934021233

KEYWORDS: capillary electrophoresis / mass spectrometry / on-line solid phase extraction / microRNA

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ABSTRACT In this paper, an on-line solid-phase extraction capillary electrophoresis-mass spectrometry (SPE-CE-MS) method is described for purification, preconcentration, separation and characterization of endogenous microRNA (miRNA) and their posttranscriptional modifications in serum. First, analysis by CE-MS was optimized using a standard mixture of hsa-miR-21-5p (miR-21-5p) and hsa-let-7g-5p (let-7g-5p). For SPE-CE-MS, a commercial silicon carbide (SiC) resin was used to prepare the microcartridges. Under the optimized conditions using standards, microcartridge lifetime (>25 analyses) and repeatability (2.8% RSD for migration times; 4.4 and 6.4% RSD for miR-21-5p and let-7g-5p peak areas, respectively) were good, the method was linear between 25-100 nmol·L-1 and limit of detection (LOD) was around 10 nmol·L-1 (50 times lower than by CE-MS). In order to analyze human serum samples, an off-line sample pretreatment based on phenol:chloroform:isoamyl alcohol (PCA) extraction was necessary prior to SPECE-MS. The potential of the SPE-CE-MS method to screen for B-cell chronic lymphocytic leukemia (CLL) was demonstrated analyzing serum samples from healthy controls and patients. MicroRNAs were only detected in CLL patients, specifically miR-21-5p and a 23-nucleotide long 5’-phosphorylated miRNA with 3’-uridylation (iso-miR-16-5p).

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MicroRNAs (miRNAs) are a class of 19–23 nucleotides long, single-stranded, non-protein coding RNAs. The function of miRNAs is to control gene expression posttranscriptionally, regulating messenger RNA (mRNA) through binding to the 3′ untranslated region (3′ UTR) of target mRNA1. miRNAs play a major role in a wide range of normal cellular processes including cell proliferation, development, and apoptosis2. Circulating miRNAs have been found in extracellular environment, including various biological fluids such as blood, saliva and urine, of both diseased and healthy people. These extracellular miRNAs are packaged in extracellular vesicles3, or associated with RNA binding proteins4,5, leading to high stability and resistance to endogenous RNAse activity, extreme temperature and pH6. Deregulation of miRNAs has been associated with different diseases like cardiovascular diseases, diabetes, aberrant immune function and especially, cancer7. In comparison to longer biomarker molecules, miRNAs present advantages including the relatively smaller number of candidate sequences, their higher stability due to the smaller size and, thus, robustness of detection. Furthermore, they are detectable in accessible biofluids such as serum. For these reasons, miRNAs are great candidates as minimally invasive biomarkers for early disease diagnosis, prognosis, and treatment outcome of different diseases8. In this sense, miR-21-5p is one of the most frequently upregulated miRNAs in solid tumors and its levels have been associated with the relapse-free survival9,10. Several analytical methods have been developed for detection of miRNA and the most commonly used are northern blotting11, microarrays12 and a two-step procedure of modified reverse transcription polymerase chain reaction followed by quantitative PCR (RT-qPCR)13. The last method is very sensitive, but it is based on indirect detection of complimentary DNA. Furthermore, the above mentioned methods are not capable of



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detecting the post-transcriptional modifications of miRNAs such as 5’-end phosphorylation and dephosphorylation, or unexpected isoforms with differing ends due to trimming or nucleotide additions14. These modifications have been reported to affect stability of miRNA and to be a mechanism for regulation of miRNA activity15. Thus, development of direct analytical methods able to obtain information regarding posttranscriptional modifications of miRNA is of great interest. In this respect, capillary electrophoresis-mass spectrometry (CE-MS) is a highly efficient technique for separation and characterization of biomolecules, generally applied in the field of proteomics and metabolomics, and less in genomics because of the emergence of next generation sequencing techniques (NGS)16–20. CE-MS has rarely been used for analysis of small RNAs although it allows direct, label-free, multiplex analysis and precise mass identification of post-transcriptional modifications of miRNA21. Small RNAs can be isolated from biological fluids using commercially available kits and then analyzed by CE-MS. However, the small injection volumes typical of CE compromise the concentration sensitivity. The use of highly selective and sensitive mass spectrometers is often not enough for robust detection, consequently, CE-MS has been often combined with different electrophoretic and chromatographic techniques for the on-line preconcentration of the target analytes, such as sample stacking, isotachophoresis, or on-line solid phase extraction (SPE-CE)22–27. In SPE-CE, a microcartridge with an appropriate sorbent is inserted near the inlet of the separation capillary to purify and preconcentrate the target analytes from a large volume of sample. SPE-CE-MS has been extensively applied for the analysis of small molecules (drugs, metabolites, etc), peptides and proteins, using the silica or polymeric sorbents typically used in off-line SPE (e.g. C18)28–30 because of the versatility of these sorbents and commercial availability. The use of other sorbents for improved selectivity, such as immunoaffinity


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sorbents, has also been studied with good results, although the very specific conditions required for the extraction and compatibility with on-line MS detection31–33. In recent years, different commercially available kits, based on SPE spin microcolumns used in combination with liquid-liquid extraction and precipitation, have been developed for miRNA isolation and purification from biological fluids. These multi-step sample pretreatments are widely used prior to PCR-based analytical methods due to the excellent recoveries and simplicity of usage34–36. However, to the best of our knowledge, it has not yet been explored the on-line coupling of these SPE methods to a high performance separation technique with MS detection, which may reduce manual handling and increase analysis throughput while obtaining valuable structural information. In this paper, for the first time, a SPE-CE-MS method for purification, preconcentration, separation and untargeted multiplex analysis of miRNAs from serum samples is described. The SPE-CE-MS analysis of miRNA was optimized using standards and the figures of merit were compared to those of a previously developed sample stacking CE-MS method21. Subsequently, the applicability of the developed SPE-CE-MS method was demonstrated analyzing serum samples from healthy controls and chronic lymphocytic leukemia (CLL) patients - the most common type of leukemia in adults37.



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Experimental section

Materials and reagents

All the chemicals used in the preparation of background electrolytes (BGEs) and solutions were of analytical reagent grade or better. Acetic acid (glacial), ammonium hydroxide (25%), guanidinium chloride (≥99.0%), ortho-phosphoric acid (85%) and sodium hydroxide (≥99.0%) were purchased from Merck (Darmstadt, Germany). Acetonitrile (LC-MS), ethanol 96% and methanol (LC-MS) were supplied by Panreac AppliChem (Barcelona, Spain). Propan-2-ol (LC-MS) was purchased from Scharlau (Barcelona, Spain). Ammonium acetate (NH4Ac) (≥99.999%), ammonium bicarbonate (for LC-MS), chloroform (≥99.0%) and phenol:chloroform:isoamyl alcohol mixture (PCA, 25:24:1) were supplied by Sigma-Aldrich (Madrid, Spain). TRIzol® Reagent was purchased from Thermo Fisher Scientific (Barcelona, Spain). Synthetic miRNA hsa-miR-21-5p (miR-21-5p), hsa-let-7g-5p (let-7g-5p) and hsa-miR-16-5p with 3’uridylation (iso-miR-16-5p) were purchased from IDT (Coralville, IA, USA).

Electrolyte solutions, sheath liquid, miRNA standards and serum samples.

All the solutions were stored in plastic bottles to minimize cation adduct formation due to the Na+ and K+ leaching from glass bottles and were degassed for 10 min by sonication before use. The optimized BGE for CE-MS and SPE-CE-MS separation contained 25 mM NH4Ac, adjusted to pH 8.0 with ammonium hydroxide. The BGE was passed through a 0.20 µm nylon filter (Macherey-Nagel, Düren, Germany). The sheath liquid solution consisted of a mixture of 80:20 v/v propan-2-


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ol:water with 2 mM NH4Ac and was delivered at a flow rate of 3.3 µL·min-1 by a KD Scientific 100 series infusion pump (Holliston, MA, USA). Stock solutions (100 µmol·L-1) of each miRNA were stored in a freezer at -20ºC. The concentration of these standard solutions was confirmed by measuring the absorbance at 260 nm (10 mm path length) using a NanodropTM spectrophotometer (Thermo Fisher Scientific, Rockford, IL, USA). Working standard solutions were prepared from the stock solution by dilution in water and were stored in the fridge at 5ºC when not in use. Human serum samples and data from donors were provided by the INCLIVA BioBank (PT13/0010/0004; B.0000768 ISCIII), integrated in the Valencian Biobanking Network and the Spanish National Biobanks Network. They were processed following standard operating procedures with the appropriate approval of the Ethical and Scientific Committees. Three healthy donor serum samples and three CLL serum samples from patients with disease stage IV were analyzed. All samples corresponded to males and females aged between 75 and 85 years. Serum aliquots were stored in a freezer at -80ºC. Healthy and CLL samples were thawed and separately pooled before the analysis.

Apparatus

pH measurements were made with a Crison 2002 potentiometer and 52-03 electrode (Crison Instruments, Barcelona, Spain). Centrifugal filtration was carried out at 25ºC in a cooled Rotanta 460 centrifuge (Hettich Zentrifugen, Tuttlingen, Germany).



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CE-MS

Fused silica capillaries were supplied by Polymicro Technologies (Phoenix, AZ, USA). All CE-MS experiments were performed in a 7100 CE coupled with an orthogonal G1603A sheath-flow interface to a 6220 oa-TOF LC/MS spectrometer (Agilent Technologies, Waldbronn, Germany). ChemStation and MassHunter softwares (Agilent Technologies) were used for CE and TOF mass spectrometer control, data acquisition, integration and mass spectrum deconvolution. The TOF mass spectrometer was operated in negative electrospray ionization mode (ESI–) and the optimized parameters are presented in the Supporting Information. Separations were performed at 15°C in a 72 cm long (LT) × 75 µm id × 365 µm od capillary. All capillary rinses were performed at high pressure (930 mbar). New capillaries, or between workdays, were both flushed with 1 M NaOH (15 or 5 min), water (15 or 10 min) and BGE (30 or 15 min) off-line to avoid the unnecessary contamination of the MS system. Samples were hydrodynamically injected at 50 mbar for 10 s and separation voltage of +20 kV (normal polarity, cathode in the outlet) was applied. The autosampler was kept at 10°C using an external water bath (Minichiller 300, Peter Huber Kältemaschinenbau AG, Offenburg, Germany). Between consecutive runs, the capillary was conditioned flushing with water (2 min) and BGE (2 min).

Sample stacking CE-MS

An electrophoretic preconcentration CE-MS method for the analysis of miRNA described elsewhere21 was adapted to our specific CE-MS set-up. Separations were


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performed at 15°C in a 158 cm long (LT) × 50 µm id × 365 µm od capillary with a BGE of 25 mM NH4Ac, adjusted to pH 6.0 with acetic acid. All capillary rinses were performed at high pressure (930 mbar). Before the injection, the capillary was rinsed with water (10 min), and BGE (3 min). Samples were injected at 930 mbar for 4 min (≈ 2.2 µL, using the Hagen–Poiseuille equation38) and then, the long plug of sample was pushed with BGE for 50 s (0.5 µL). Separation was performed at +20 kV.

SPE-CE-MS

The silicon carbide (SiC) resin was obtained from the commercial microRNA purification kit spin columns of Norgen Biotek Corp. (Thorold, Canada). Construction of the microcartridge or analyte concentrator was carried out as described elsewhere with little modifications31,39,40. The inlet end was prepared connecting the microcartridge (0.7 cm LT × 250 µm id × 365 µm od capillary) with a plastic sleeve to a previously conditioned inlet capillary (7.5 cm LT x 75 µm id × 365 µm od). The microcartridge was completely filled by vacuum with the SiC particles. Another plastic sleeve was connected to the microcartridge and a small piece of cotton (approx. 1 mm) was introduced in the plastic tube before connecting the separation capillary (64.5 cm LT x 75 µm id × 365 µm od). This cotton frit prevented leaking of the SiC particles, which promoted current instability or breakdown and poor reproducibility. Samples were hydrodynamically introduced at 930 mbar for 5 min (60 µL). A final flush for 40 s with BGE eliminated non-retained molecules and equilibrated the capillary before the electrophoretic separation. All these steps were performed by switching off the nebulizer gas and the ESI capillary voltage to prevent contaminants entrance into the MS. Then, both were switched on, a small volume of eluent with 60%



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v/v of acetonitrile (ACN) was injected at 50 mbar for 10 s (50 nL), and separation was conducted at +20 kV at 15°C, applying 25 mbar of pressure to compensate for microcartridge counter-pressure. Between consecutive runs, the capillary was rinsed for 4 min with water. Before SPE-CE-MS, serum samples were extracted with PCA or TRIzol® Reagent both followed by chloroform extraction. The detailed procedure is presented in the Supporting Information.

Quality parameters The details regarding estimation of the LOD, LOQ, repeatability of migration time and peak area, linearity, and microcartridge lifetime in CE-MS or SPE-CE-MS are given in the Supporting Information.

Results and discussion

CE-MS

CE-MS was optimized by analyzing standard solutions of the synthetic miR-215p and let-7g-5p miRNAs (Table S-1A). Most MS studies on nucleic acids use ESI-, because it yields a better signal for such acidic compounds21,41,42. However, since the phosphate groups of the miRNA have cation-binding sites, clusters corresponding to Na+ and K+ adducts are typically detected in the mass spectrum, decreasing the signal of the expected [M-nH]n- molecular ions. In order to increase the sensitivity of the detection of miRNA in ESI– and to decrease the cation adduct formation, several BGE and sheath liquid compositions were tested. In general, an effective method to minimize alkali adduct formation is the addition of 11 ACS Paragon Plus Environment

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organic bases or ammonium salts43. In our case, appropriate results were obtained with BGEs of ammonium acetate. Different BGE concentrations and acidities were tested at 10, 25 and 50 mM NH4Ac and pH 6.0 and 8.0, adjusted with acetic acid or ammonium hydroxide, respectively. The lowest adduct formation and highest sensitivity were obtained with a BGE of 25 mM NH4Ac at pH 8.0, in contrast to a previous study that reported a BGE of 25 mM NH4Ac at pH 6.0 for the analysis of miRNA by sample stacking CE-MS21. This BGE was always freshly prepared in plastic tubes to minimize cation adduct formation due to the Na+ and K+ leaching from glass bottles. Different hydroorganic mixtures were also tested as sheath liquid, including propan-2-ol, methanol and acetonitrile at 60 and 80% v/v. The highest sensitivity for miRNA detection was obtained with 80% v/v propan-2-ol (Figure S-1A). NH4OH 0.5% v/v was also added to this mixture to improve miRNA ionization but sensitivity was lower than with ammonium salts as additives. The optimized sheath liquid contained 80% v/v propan-2-ol and 2 mM NH4Ac, as in the sample stacking CE-MS method indicated before21, showing better results than with 1, 5 and 10 mM NH4Ac. As an example, Figure 1 shows the Extracted Ion Electropherogram (EIE) and the mass spectrum of a 5000 nmol·L-1 standard mixture of miR-21-5p and let-7g-5p in the optimized conditions. Both miRNA co-migrated, but showed molecular ions with different m/z values due to the molecular mass differences. The most abundant ion was the [M-5H]5- and some Na+ and K+ adducts were still detected ([M-6H+Na]5- and [M6H+K]5-). Under these conditions, the LOD of the CE-MS method was 500 nmol·L-1 for both miRNAs.

Sample stacking CE-MS adaptation



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Before exploring SPE-CE-MS, we adapted the sample stacking CE-MS method previously described for the analysis of miRNAs to the specific instrumental set-up used in this study21. Sample stacking preconcentration during CE-MS was achieved by dissolving the sample in water, which had lower conductivity than the BGE. A large volume of sample was injected hydrodynamically in the capillary between two plugs of BGE. When separation voltage was applied, focusing and preconcentration of the ions happened at the narrow zone of the interface between the sample solution and the BGE because of a change in electrophoretic velocity due to a difference in the conductivities and, hence, in the electric field. As an example, Figure 2 shows the analysis of a 250 nmol·L-1 standard mixture of miR21-5p and let-7g-5p in the adapted conditions. As expected from the results of the previous study21, migration times were five times higher using sample stacking than by conventional CE-MS (Figure 2A) and the mass spectrum showed the same ion clusters as by CE-MS (compare Figures 2B and 1B). The LOD of the sample stacking CE-MS method adapted to our instrumental set-up was 50 nmol·L-1, 10 times lower than by CEMS, 500 nmol·L-1. A slightly lower LOD (20 nmol·L-1) was obtained by sample stacking CE-MS in the original study21, probably because of the better sensitivity of the mass spectrometer used in that case. Sample stacking allows a remarkable preconcentration factor, and in that study it was demonstrated that it was possible to analyze miRNA from cancer serum samples21. As an alternative, in this study we investigated SPE-CE-MS to further enhance sensitivity, while reducing manual handling and increasing analysis throughput.

SPE-CE-MS optimization


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The SiC resin from the spin microcolumns of the microRNA isolation kit from Norgen was used to prepare the on-line SPE-CE microcartridges. The literature provided by the manufacturer cited better recoveries and linearities on miRNA purification than with silica-based or a traditional phase separation methods44. Another important aspect of the SiC resin was the small particle size, which facilitated packing the on-line microcartridges. The starting point for optimization of the SPE-CE–MS methodology was the procedure recommended by the manufacturer, as well as on other procedures applied for extraction of hydrophilic analytes, such as protein glycans, with graphitic carbon sorbents45,46. In SPE-CE-MS, after sample loading, the microcartridge located at the capillary inlet needed to be washed to eliminate non-specifically retained molecules, and also filled with BGE before the elution, separation and detection. In preliminary studies using the optimized BGE for CE-MS, 25 mM NH4Ac (pH=8.0), some miRNA elution was detected during the washing and filling step with BGE (930 mbar, 2 min). This miRNA leakage was detrimental to achieving the optimal sensitivity. Different strategies were explored, but it was found through those studies that changes in NH4Ac concentration or pH, and also ammonium phosphate and bicarbonate BGEs had poorer performance. Several modifications in the pressure and duration of the washing and filling step were, therefore, evaluated while minimizing the total volume of the flushed solution (4.0 µL, approximately 1.3 capillary volumes). This step was performed at low pressure (100 mbar, 360 s)38, but lower sensitivities than those at high pressure (930 mbar, 40 s)38 were obtained. Several other alternatives were explored including the use of guanidinium chloride (0.97) between 25 and 100 nmol·L-1 for both miRNAs. When loading a concentration of 150 mM, the sorbent was saturated and there was no the expected increase in the peak areas. The LOD was 10 nmol·L-1, which was an improvement of 50 and 5 times compared to CE-MS and sample stacking CE-MS methods, respectively. The LOQ was 25 nmol·L-1. The lifetime of the microcartridges was around 25 analyses. As an example, Figure 4 shows the SPECE-MS analysis of a 50 nmol·L-1 miR-21-5p and let-7g-5p standard mixture in the optimized conditions. In comparison to CE-MS (Figure 1), migration times were similar, number of theoretical plates (N) slightly higher and the same ions were detected in the mass spectrum.

MicroRNA detection in cancer serum

The SPE-CE-MS method optimized with standards was applied to the analysis of biological fluids. Considering that miRNA levels in serum and plasma samples are similar6 and that serum samples are more abundant in many clinical sample repositories, serum samples were selected for miRNA profiling. Extracellular circulating miRNAs are found in serum of both diseased and healthy people, but at differing levels of concentration. miRNAs are resistant to endogenous RNAse activity, as well as extreme pH and temperature6,8 because they are contained within microvesicles or in protein and lipoprotein complexes9. For this reason, no miRNAs were detected when serum samples were loaded without any off-line pretreatment (e.g. addition of lysis buffer or phenol solution). In addition, current instability and breakdown during the electrophoretic separation was observed because of saturation of the sorbent with the sample matrix components due to non-specific retention. Therefore, an off-line sample pretreatment



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was required to inhibit RNase activity, dissociate the nucleocomplexes, precipitate proteins and extract the miRNAs. Two extraction procedures were tested based on TRIzol® and PCA, in both cases followed by chloroform extraction. In addition, it was necessary to desalt the extracts since retention of the miRNAs was compromised due to the high abundance of salts. Drop dialysis was performed because it is a simple, rapid and highly effective method to remove salts from small volume samples47,48. The sample pretreatments were optimized with healthy control serum samples spiked with the standard miRNAs miR-21-5p and let-7g-5p. The PCA extraction procedure was selected because of the limited compatibility of TRIzol® Reagent with MS, due to the presence of high concentration of chaotropic salts (i.e. guanidinium thiocyanate). In the optimized conditions, the sample pretreatment recoveries were high, approximately 85%, calculated by comparison of the SPE-CE-MS analysis for a 50 nmol·L-1 standard solution and a healthy control serum sample spiked at the same concentration. Consecutive analyses were repeatable in terms of migration times and peak areas (%RSD (n=3) 3.9 and 7.6% for miR-21-5p and 3.9 and 4.1% for let-7g-5p, respectively, for a healthy control serum sample spiked at 50 nmol·L-1). Furthermore, the lifetime of the microcartridges and LOD were similar as with standards (around 20 analyses and 10 nmol·L-1, respectively). With the optimized sample pretreatment and SPE-CE-MS method, in concordance with the previous study using the sample stacking CE-MS method21, no endogenous miRNAs were detected in (unspiked) healthy control serum samples (Figures 5A and B), because of the extremely low abundance of miRNA in this type of samples. It is well known that some miRNAs are up-regulated in serum from patients of different types of cancer9,49–52. CLL is a cancer of the B-lymphocytes and is the most common type of leukemia in adults37. CLL derives from a combination of genetic


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(chromosomal abnormalities, gene mutations) as well as epigenetic (altered microRNA expression, DNA methylation) modifications53. Specific miRNA profiles have been associated with CLL progression, prognosis and drug resistance54. With SPE-CE-MS, in the analyzed CLL serum sample, two miRNAs were clearly detected at concentrations near the LODs (the EIEs and the mass spectrum are shown in Figures 5C and D, respectively). The two miRNAs were tentatively identified using miRBase, which is a comprehensive

database

of

miRNA

sequences

and

expression

(http://www.mirbase.org)55. The current version (miRBase 21) indexes 28,645 miRNA entries and about 2,000 sequences for humans. An screening of the most abundant miRNA species in CLL plasma and cell samples reported in the literature (Table S-2)56– 59

was performed, taking into account possible post-transcriptional modifications. The

most likely possible candidates were miR-21-5p and miR-16-5p with 3′-uridylation (iso-miR-16-5p) (Table S-1B). The analysis of a spiked CLL sample allowed confirmation of the identity of the two miRNAs (Figures 5E and 5F). The detection of these two miRNA was in good agreement with the results reported for similar samples by sample stacking CE-MS and RT-qPCR21,60. miR-21-5p was the first discovered serum miRNA biomarker in serum of CLL patients10, it is a representative oncogenic miRNA and the overexpression of miR-21-5p as well as miR-16-5p in CLL has been previously reported21,60. In the future, the identities of the detected miRNAs could be unambiguously confirmed by high resolution tandem MS analysis with a hybrid mass spectrometer.

Concluding remarks



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We have developed an on-line SPE-CE-MS method for purification, preconcentration, separation and characterization of endogenous microRNAs and their post-transcriptional modifications in serum. Under the optimized conditions with standards, microcartridge lifetime (around 25 analyses) and repeatability (2.8 and 85% and performance of the SPE-CE-MS method similar as with standards. Under the optimized conditions, it was possible to distinguish healthy controls and CLL patients, because only in the diseased group two endogenous miRNAs were detected at very low concentration. The results achieved show that SPE-CE-MS can be regarded as a promising technique for direct, high-throughput, sensitive, multiplex analysis capable of preconcentration, quantification, and characterization of miRNA. The method also provides the unique structural information necessary for understanding the post-transcriptional regulation of miRNA function, and can be used in the discovery of new miRNA biomarkers of cancer and other diseases in non-invasive biological fluids such as serum or plasma.

Supporting Information

Optimized MS parameters, serum sample extraction, quality parameters, characteristics and molecular masses of the analyzed miRNAs, study of the sheath liquid in CE-MS and the eluent and linearity in SPE-CE-MS and the list of miRNAs used for the screening of the CLL serum sample.


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Acknowledgements This study was supported by a grant from the Spanish Ministry of Economy and Competitiveness (CTQ2014-56777-R). Roger Pero-Gascon acknowledges the Spanish Ministry of Education, Culture and Sport for a FPU (Formación del Profesorado Universitario) fellowship. Maxim V. Berezovski thanks the NOVA DOMUS – CHEMEDPHO project funded by the Erasmus Mundus Action II of the European Commission for a travel grant. We want to particularly acknowledge the patients and the INCLIVA BioBank (PT13/0010/0004; B.0000768 ISCIII) integrated in the Valencian Biobanking Network and the Spanish National Biobanks Network for providing the serum samples. Authors thank Ryan Girgrah and Emil Zaripov for English grammar correction.

The authors have declared no conflict of interest.

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Figure 1. CE-MS for a 5000 nmol·L-1 miR-21-5p and let-7g-5p standard mixture. A) Extracted Ion Electropherogram (EIE) and B) Mass spectrum. The number of theoretical plates (N) was calculated as N=5.54*(tm/w1/2)2 (tm=migration time and w1/2= width at half the peak height).

Figure 2. Sample stacking CE-MS for a 250 nmol·L-1 miR-21-5p and let-7g-5p standard mixture. A) Extracted Ion Electropherogram (EIE) and B) Mass spectrum. The number of theoretical plates (N) was calculated as N=5.54*(tm/w1/2)2 (tm=migration time and w1/2= width at half the peak height).



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Figure 3. Plot of peak area of the eluted miR-21-5p (♦) and let-7g-5p (□) vs loading time at 930 mbar (35 nmol·L-1 standard mixture).

Figure 4. SPE-CE-MS for a 50 nmol·L-1 miR-21-5p and let-7g-5p standard mixture. A) Extracted Ion Electropherogram (EIE) and B) Mass spectrum. The number of theoretical plates (N) was calculated as N=5.54*(tm/w1/2)2 (tm=migration time and w1/2= width at half the peak height).


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Figure 5. SPE-CE-MS for human serum samples: Extracted Ion Electropherogram (EIE) and mass spectrum of A and B) a healthy control, C and D) a CLL patient and E and F) a CLL patient (spiked with 50 nM miR-21-5p and 50 nM iso-miR-16-5p). No endogenous miRNAs were detected in the control.



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For Table Of Contents (TOC) only


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190x254mm (300 x 300 DPI)



190x254mm (300 x 300 DPI)


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190x254mm (300 x 300 DPI)



190x254mm (300 x 300 DPI)


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190x254mm (300 x 300 DPI)


Analysis of circulating microRNAs and their post-transcriptional

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